1. Technical Field
The present invention relates to a temporal focusing-based multiphoton excitation fluorescence microscopy system capable of tunable-wavelength excitation and an excitation wavelength selection module thereof. More particularly, the invention relates to a fluorescence microscopy system for examining diverse bio-tissues and the configuration of the system's wavelength selection module.
2. Description of Related Art
Multiphoton excitation (MPE) fluorescence microscopes can be used in the biomedical field. Generally, the wavelength of the excitation light is selected according to the fluorescent dye with which to label a specimen, and the labeled specimen is scanned to obtain fluorescence images for display and analysis. In multifluorophore imaging, however, MPE fluorescence microscopy with a fixed-wavelength pulse laser illumination configuration is insufficient to perform efficient excitation of fluorophores with different two-photon absorption spectra.
Many fluorescence proteins and fluorophores used in biomedical studies have demonstrated the specific spectra of multiphoton absorption. The highly effective multiphoton excitation of fluorophores provides superior signal-to-noise ratio (SNR) MPE imaging, where SNR is critical to the imaging depth of three-dimensional biospecimens. Although fluorescent excitation of two different fluorophores with overlapping two-photon absorption spectra can be achieved using conventional MPE fluorescence microscopes with a single excitation wavelength, the selection of fluorophores for biomedical applications is restricted.
In spatial-focusing-only MPE microscopy where galvo scanners are used to map MPE fluorescence images, matching the maximum multiphoton absorption wavelengths of different fluorophores is straightforward through adjustment of the incident wavelength of ultrafast pulse lasers. In a temporal focusing-based AVE fluorescence microscopy system, however, adjusting the incident wavelength in real time to obtain the optimum efficiency of MPE for higher image quality is difficult because of the use of a diffraction device, which separates the frequencies of incident pulses at different diffraction angles.